Bacillus aryabhattai and application thereof

Abstract

The invention relates to the field of microorganisms, and discloses a strain of bacillus aryabhattai, which is characterized in that the preservation number of the bacillus aryabhattai is CCTCC (China Center for Type Culture Collection) NO: M 2024282. The bacillus aryabhattai provided by the invention can inhibit the activity of the pathogenic bacterium fusarium oxysporum f.sp.cubense of banana wilt, so that the banana wilt can be prevented and treated, and in addition, the bacillus aryabhattai also has a remarkable growth promoting effect on banana plants.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microorganisms, in particular to the genus Priestia and its application. BACKGROUND

[0002] Banana (Musa spp.) is an important fruit in the world's tropical and subtropical regions, and there are more than 130 countries in the world growing bananas. Banana wilt is a devastating disease caused by the vascular bundle of banana plants infected by Fusarium oxysporum f. sp. cubense (Foc) 4 physiological race (TR4). The symptoms of banana wilt are wilting and vascular discoloration and rotting of the diseased plant: the initial stage of the diseased leaf edge is yellow, and gradually converges to the leaf vein; at the same time, the diseased leaf wilts rapidly, and the whole leaf is hanging on the side of the pseudostem; then, all the leaves appear yellow, wilt, and hang down from bottom to top, and the top leaves cannot be pulled out; the cross section of the diseased plant pseudostem shows yellow or red-brown spots, and the closer to the stem base, the deeper the color of the diseased vascular bundle, and the root is black-brown and gradually rots, with a special odor. The pathogen of the disease can survive in the soil for several years, and the yield is reduced by more than 20%, and the severe field even causes absolute yield loss. Banana wilt has been reported in all banana production areas in China, which has caused serious impact on the banana industry.

[0003] The main prevention and control measures for banana wilt include breeding disease-resistant varieties, chemical agents, crop rotation, biological control, molecular genetic improvement, etc. Based on the poor yield of disease-resistant varieties and the unsatisfactory effect of chemical agents, safe, efficient and environmentally friendly biological control technology has gradually become one of the effective measures for the prevention and control of banana wilt. At present, there are still few strains that can prevent and control banana wilt without affecting the growth of banana, and even promote the growth of banana. The lack of candidate strains for preventing and controlling banana wilt requires the search for new antagonistic bacteria. SUMMARY

[0004] The purpose of the present application is to overcome the problems existing in the prior art, and to provide a strain of Priestia arya bhattai and its application, which can effectively prevent and control banana wilt and promote the growth of banana.

[0005] In order to achieve the above-mentioned purpose, the first aspect of the present application provides a strain of Priestia arya bhattai, characterized in that the preservation number of the strain of Priestia arya bhattai is CCTCC NO: M2024282.

[0006] The second aspect of the present application provides a bacterial agent, characterized in that the bacterial agent contains the strain of Priestia arya bhattai.

[0007] The application provides application of the Bacillus aryabhattai or the microbial agent in inhibition of Fusarium oxysporum f.sp.cubense.

[0008] The application provides a method for preventing and treating banana wilt and / or promoting growth of banana plants, and the method comprises: applying the fermentation liquor of the Bacillus aryabhattai or the microbial agent to the banana plants.

[0009] The Bacillus aryabhattai provided by the application is an endophytic bacterium which can inhibit the activity of Fusarium oxysporum f.sp.cubense, the pathogenic bacterium of banana wilt, thereby preventing and treating banana wilt, reducing the severity of banana wilt, reducing the symptoms of yellowing of leaves, wilting of leaves, browning and rotting of corms and the like caused by banana wilt, and additionally having a significant growth promoting effect on banana plants, in particular, increasing plant height, increasing the number of leaves, increasing the diameter of pseudostems, and increasing the fresh weight of aboveground and underground parts.

[0010] The Bacillus aryabhattai provided by the application can enrich the resources of biocontrol bacteria for banana wilt, provide a certain theoretical basis for further field development and application of the banana growth-promoting and disease-resistant strain, and provide a reference for development of biological bacterial manure and biological bacterial agent and other biological products for biological prevention and control.

[0011] Biological preservation

[0012] The Bacillus aryabhattai (Priestia aryabhattai) provided by the application was preserved in the China Center for Type Culture Collection (CCTCC) (address: Wuhan University, Wuhan, China, postcode: 430072) on January 29, 2024 (the abbreviation of the preservation unit is CCTCC), and the preservation number is CCTCC NO:M2024282. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 YN2302 is the antibacterial effect of banana wilt pathogen, A is the PDA plate just after inoculation of YN2302, and B is the PDA plate after inoculation of YN2302 for 7 days.

[0014] Figure 2 is the growth condition of banana wilt pathogen of the PDA culture medium inoculated only with the pathogenic bacterium to be tested.

[0015] Figure 3 is the growth condition of banana wilt pathogen of YN2302.

[0016] Figure 4 is the growth-promoting condition of banana plants of YN2302. DETAILED DESCRIPTION

[0017] The endpoints of the ranges and any values disclosed herein are not limited to the precise values recited as the exact dimensions are not critical to the invention. Any numeric range recited is intended to include all values from the lower value to the upper value, inclusive of both values, and to also include any value approximately or about the value. For numeric ranges, the endpoints are combined with the single points to form new numeric ranges, which are to be construed as being specifically disclosed.

[0018] The first aspect of the present application provides a strain of Priestia aryabhattai, characterized in that the preservation number of the strain of Priestia aryabhattai is CCTCC NO: M 2024282.

[0019] The strain of Priestia aryabhattai provided by the present application is isolated from the healthy banana root system of a banana wilt plot in Lianghe County, Dehong Prefecture, Yunnan Province, and has the following morphological characteristics: the colony on the plate medium is round, slightly yellow, moist, and has wrinkles and a mucous appearance. It is a gram-positive bacterium, has a rod-shaped body, can move, and can produce endospores.

[0020] The second aspect of the present application provides a microbial agent, characterized in that the microbial agent contains the strain of Priestia aryabhattai.

[0021] In the present application, the preparation method of the microbial agent can refer to the conventional preparation method in the art, which will not be described here.

[0022] In the present application, the form of the microbial agent can be the conventional form of the microbial agent in the art, such as a solid, liquid or semi-solid form.

[0023] In the present application, the number of viable cells in the microbial agent can be selected within a wide range, as long as it meets the requirements of the relevant standards. For example, in a liquid microbial agent, the content of viable cells in the microbial agent is 1x10 8 CFU / mL or more.

[0024] The third aspect of the present application provides an application of the strain of Priestia aryabhattai or the microbial agent in inhibiting Fusarium oxysporum f.sp.cubense.

[0025] In the present application, in order to improve the inhibition rate of Fusarium oxysporum f.sp.cubense, preferably, for every 4x10 7 CFU of Fusarium oxysporum f.sp.cubense, the amount of the strain of Priestia aryabhattai is not less than 1x10 8 CFU, more preferably 8x10 8 CFU to 1x10 10 CFU, further preferably 1x10 9 CFU to 6x109 CFU.

[0026] The fourth aspect of the present application provides a method for preventing and treating banana fusarium wilt and / or promoting the growth of banana plants, the method comprising: applying the Bacillus aryabhattai or the fermentation broth of the bacterial agent to banana plants. The method provided by the present application can reduce the severity of banana fusarium wilt, reduce the symptoms of banana fusarium wilt such as leaf yellowing, leaf wilting, corm browning and rotting, and also promote the growth of banana, especially in the growth of banana plants, which can increase the plant height, increase the number of leaves, increase the diameter of the pseudostem, and increase the fresh weight of the aboveground and underground parts.

[0027] In the present application, preferably, the preparation method of the fermentation broth comprises: inoculating the Bacillus aryabhattai into a culture medium for culture.

[0028] In the present application, the liquid culture medium is not limited, and can be used for the smooth fermentation of Bacillus aryabhattai, and is preferably NA culture medium. In some preferred embodiments, the liquid culture medium contains: preferably, the weight ratio of the yeast extract, peptone, beef extract and glucose is 1:(5-14):(1-5):(8-25); preferably, the total weight of the yeast extract, peptone, beef extract and glucose accounts for 1-5% of the weight of the culture medium, and more preferably 2-3%. In the present application, when the culture medium contains yeast extract, peptone, beef extract and glucose, the obtained fermentation broth can further reduce the severity of banana fusarium wilt, reduce the symptoms of banana fusarium wilt such as leaf yellowing, leaf wilting, corm browning and rotting, and also promote the growth of banana, especially in the growth of banana plants, which can increase the plant height, increase the number of leaves, increase the diameter of the pseudostem, and increase the fresh weight of the aboveground and underground parts.

[0029] In the present application, preferably, the total nitrogen content in the dry base of the yeast extract per gram is more than 5% by weight.

[0030] In the present application, preferably, the content of amino acid nitrogen in the dry base of the yeast extract per gram is less than 6% by weight, for example, it can be 6% by weight, 5% by weight, 4% by weight, 3% by weight, 2% by weight, 1% by weight, 0% by weight, and any range and any value in the range composed of any of the above values.

[0031] In the present application, preferably, the pH of the liquid culture medium is 6-8.

[0032] In the present application, preferably, the culture conditions include: time of 2-5 days (for example, it can be 2 days, 2.5 days, 3 days, 3.5 days, 4 days, 4.5 days, 5 days, and any range and any value in the range composed of any of the above values), temperature of 35-40℃ (for example, it can be 35℃, 36℃, 37℃, 38℃, 39℃, 40℃, and any range and any value in the range composed of any of the above values).

[0033] In the present application, in order to make Bacillus aryabhattai better fermentation, the culture process can be oscillated, and the oscillation rate is 200-300 r / min.

[0034] In the present application, before the Bacillus aryabhattai is inoculated into the liquid culture medium for culture, it can be activated first, and the activation method is to inoculate the Bacillus aryabhattai into a solid culture medium. After the colony grows, it is inoculated into the liquid culture medium.

[0035] In the present application, the solid culture medium is not limited, and it can only make the Bacillus aryabhattai activate smoothly, preferably, it is NA solid culture medium, more preferably, the solid culture medium contains yeast extract, peptone, beef extract and glucose, and the weight ratio of the yeast extract, peptone, beef extract and glucose is preferably 1:(5-14):(1-5):(8-25), more preferably, the weight ratio of the yeast extract, peptone, beef extract and glucose is 1:(6-10):(2-4):(12-18). The total weight of the yeast extract, peptone, beef extract and glucose accounts for 1-5% of the weight of the culture medium, more preferably 1-2%, and the pH of the solid culture medium is preferably 6-8. Further preferably, the culture conditions of the solid culture medium include: time of 0.5-2 days, temperature of 35-40℃. The glucose can be provided by glucose containing crystal water or anhydrous glucose, and the glucose content in the glucose containing crystal water and anhydrous glucose meets the above range.

[0036] In the present application, the application amount of the Bacillus aryabhattai and the application amount of the bacterial agent containing the Bacillus aryabhattai can be determined according to the degree of fusarium wilt of banana plants. In some embodiments of the present application, preferably, the application amount of the Bacillus aryabhattai is not less than 1×10 8 CFU / plant / time, preferably 8×10 8 CFU / plant / time to 1×10 10 CFU / plant / time (for example, it can be 8×10 8 CFU / plant / time, 9×10 8 CFU / plant / time, 1×10 9 CFU / plant / time, 2×10 9 CFU / plant / time, 3×10 9CFU / time, 4×10 9 CFU / time, 5×10 9 CFU / time, 6×10 9 CFU / time, 7×10 9 CFU / time, 8×10 9 CFU / time, 9×10 9 CFU / time, 1×10 10 CFU / time and any range of the above values ​​and any value within the range), more preferably 1×10 9 CFU / plant / time up to 6×10 9 CFU / plant / time; or

[0037] The amount of fermentation broth used in the bacterial agent is such that the amount of *Bacillus argentea* is not less than 1 × 10⁻⁶. 8 CFU / plant / dose, preferably 8×10 8 CFU / plant / time up to 1×10 10 CFU / plant / dose (e.g., 8×10) 8 CFU / time, 9×10 8 CFU / time, 1×10 9 CFU / time, 2×10 9 CFU / time, 3×10 9 CFU / time, 4×10 9 CFU / time, 5×10 9 CFU / time, 6×10 9 CFU / time, 7×10 9 CFU / time, 8×10 9 CFU / time, 9×10 9 CFU / time, 1×10 10 CFU / time and any range of the above values ​​and any value within the range), more preferably 1×10 9 CFU / plant / time up to 6×10 9 CFU / strain / time.

[0038] In this invention, the application rate of *Bacillus argentea* and the application rate of the *Bacillus argentea*-containing inoculant can be determined based on the growth status of the banana plant. In some embodiments of this invention, preferably, the application rate of *Bacillus argentea* is not less than 1 × 10⁻⁶. 8 CFU / plant / dose, preferably 8×10 8 CFU / time up to 1×10 10 CFU / plant / dose (e.g., 8×10) 8 CFU / time, 9×10 8 CFU / time, 1×10 9CFU / time, 2×10 9 CFU / time, 3×10 9 CFU / time, 4×10 9 CFU / time, 5×10 9 CFU / time, 6×10 9 CFU / time, 7×10 9 CFU / time, 8×10 9 CFU / time, 9×10 9 CFU / time, 1×10 10 CFU / time and any range of the above values ​​and any value within the range), more preferably 1×10 9 CFU / plant / time up to 6×10 9 CFU / plant / time; or

[0039] The amount of fermentation broth used in the bacterial agent is such that the amount of *Bacillus argentea* is not less than 1 × 10⁻⁶. 8 CFU / plant / dose, preferably 8×10 8 CFU / plant / time up to 1×10 10 CFU / plant / dose (e.g., 8×10) 8 CFU / time, 9×10 8 CFU / time, 1×10 9 CFU / time, 2×10 9 CFU / time, 3×10 9 CFU / time, 4×10 9 CFU / time, 5×10 9 CFU / time, 6×10 9 CFU / time, 7×10 9 CFU / time, 8×10 9 CFU / time, 9×10 9 CFU / time, 1×10 10 CFU / time and any range of the above values ​​and any value within the range), more preferably 1×10 9 CFU / plant / time up to 6×10 9 CFU / strain / time.

[0040] In some preferred embodiments, for banana seedlings with 5-6 leaves, the preferred application rate of Bacillus aureus is 8 × 10⁻⁶. 8 CFU / time up to 1×10 10 CFU / plant / dose (e.g., 8×10) 8 CFU / time, 9×10 8 CFU / time, 1×10 9 CFU / time, 2×10 9CFU / time, 3×10 9 CFU / time, 4×10 9 CFU / time, 5×10 9 CFU / time, 6×10 9 CFU / time, 7×10 9 CFU / time, 8×10 9 CFU / time, 9×10 9 CFU / time, 1×10 10 CFU / time and any range of the above values ​​and any value within the range), more preferably 1×10 9 CFU / plant / time up to 6×10 9 CFU / plant / time; or

[0041] The fermentation broth of the bacterial agent is used such that the amount of *Bacillus argentea* is preferably 8 × 10⁻⁶. 8 CFU / plant / time up to 1×10 10 CFU / plant / dose (e.g., 8×10) 8 CFU / time, 9×10 8 CFU / time, 1×10 9 CFU / time, 2×10 9 CFU / time, 3×10 9 CFU / time, 4×10 9 CFU / time, 5×10 9 CFU / time, 6×10 9 CFU / time, 7×10 9 CFU / time, 8×10 9 CFU / time, 9×10 9 CFU / time, 1×10 10 CFU / time and any range of the above values ​​and any value within the range), more preferably 1×10 9 CFU / plant / time up to 6×10 9 CFU / strain / time.

[0042] The present invention will be described in detail below through embodiments. It should be understood that the following embodiments are only used to further explain and illustrate the content of the present invention by way of example, and are not intended to limit the present invention.

[0043] Unless otherwise specified, the reagents and materials used in the following examples are all commercially available products purchased from regular chemical / biological reagent or material suppliers, and all reagents are of analytical grade.

[0044] Unless otherwise specified, the operating temperature in the following embodiments is room temperature (25±5℃).

[0045] Example 1

[0046] This example is used to illustrate the obtaining, identification and cultivation of Priestia aryabhattai YN2302.

[0047] 2.1 Source of materials

[0048] The Priestia aryabhattai described in the present application is isolated from the roots of healthy banana plants in a banana wilt disease susceptible plot in Lianghe County, Dehong Prefecture, Yunnan Province, named YN2302, and preserved in the China Center for Type Culture Collection (hereinafter referred to as CCTCC) on January 29, 2024, with the preservation number CCTCC NO: M 2024282. The address of the preservation center is Wuhan University, China.

[0049] 2.2 Culture medium

[0050] PDA culture medium: glucose 15 g, agar 15 g, peeled potato 200 g, distilled water 1000 mL, pH 7.0.

[0051] NA culture medium: yeast extract 1 g, proteose peptone 8 g, beef extract 3 g, anhydrous glucose 15 g, agar powder 15 g, distilled water 1000 mL, pH 7.0. The liquid medium is obtained by not adding agar.

[0052] Yeast extract is purchased from Solarbio (Beijing Solarbio Science and Technology Co., Ltd.), product number Y8030, and the total nitrogen content in dry basis of each gram of yeast extract is more than 7.2% by weight; the content of sodium chloride in dry basis of each gram of the yeast extract is less than 5% by weight.

[0053] 2.3 Method for obtaining, identifying and cultivating

[0054] (1) Sample disinfection treatment: The roots of banana plants were pre-taken, washed with sterile water, and then surface-disinfected with 75% ethanol for 60 s, and finally washed with sterile water and air-dried.

[0055] (2) Isolation and culture: After the above-mentioned banana plant roots were disinfected, the pathogenic bacteria were isolated and cultured on NA medium under sterile conditions by using the plate streaking method, and incubated at 30°C for 1 day. After 3 times of single colony purification, the bacteria were stored in a refrigerator at 4°C for standby use.

[0056] (3) Classification and identification: The endophytic antagonistic bacteria obtained by isolation were cultured, and the culture characteristics and morphological features were observed, and 16S rDNA amplification and sequence analysis were performed.

[0057] The culture traits and morphological characteristics are observed and identified according to the Handbook of Common Bacteria System Identification; the bacterial strains obtained above are round, slightly yellow, wet, and mucous with folds on the plate culture medium; the bacterial strains are gram-positive, rod-shaped, can move, and can produce endospores.

[0058] 16S rDNA amplification and sequence analysis:

[0059] 16S rDNA amplification sequence (SEQ ID NO: 1):

[0060]

[0061] Genomic DNA was extracted with a bacterial genomic DNA extraction kit, and universal primers 27F / 1492R were used:

[0062] 5'-AGAGTTTGATCCTGGCTCAG-3'(SEQ ID NO: 2)

[0063] 5'-GGTTACCTTGTTACGACTT-3'(SEQ ID NO: 3)

[0064] The 16S rDNA gene partial sequence of the antagonistic bacteria was PCR amplified. The primer sequence was synthesized by Beijing Qikete Biotechnology Co., Ltd. Kunming Branch, and the PCR product was sequenced by Beijing Qikete Biotechnology Co., Ltd. Kunming Branch. The PCR amplification was carried out with the template. The PCR reaction system was 50 μL: TaKaRa LA-Taq 0.5 μL, DNA template 2 μL, 10x PCR Buffer 5 μL, dNTP Mixture 8 μL, 2 μL of upper and lower primers, and double distilled water 30.5 μL. The PCR program was 94°C pre-denaturation for 5 min, 94°C for 30 s, 54°C for 30 s, 72°C for 1 min, 30 cycles; 72°C extension for 7 min. After the PCR product was sequenced, the sequencing results were compared by BLAST on the NCBI website, and the MEGA7.0 software was used to construct the phylogenetic tree by the neighbor-joining method. The classification status of the isolated endophytic antagonistic bacteria was determined. The classification reference literature: Che Xiuzhu, Cai Miaoying. Common Bacterial System Identification Manual [M]. Beijing: Science Press, 2001: 43-65; Li Fudi, Yu Ziniu, He Shaojiang. Experimental Technology of Agricultural Microbiology [M]. Beijing: China Agricultural Press, 1996.

[0065] The total genomic DNA of the strain was used as a template for PCR amplification, and a PCR product of about 1.5 kb was obtained. The sequencing results were compared by Blast in NCBI, and the results showed that the sequence similarity of the strain with Priestia aryabhattai was 99%. The phylogenetic tree was constructed by MEGA5.0 software, and the results showed that the strain was most closely related to Priestia aryabhattai and clustered into a branch.

[0066] Through culture characteristics, morphological observation, physiological and biochemical tests, and 16S rDNA sequence analysis, it was determined that the strain was Priestia aryabhattai. Its code was designated as YN2302.

[0067] The culture preservation method of the Priestia aryabhattai:

[0068] Short-term culture preservation: inoculate on NA solid slant medium, after the bacteria grow well, the cotton plug is wrapped with oil paper, and then moved to the refrigerator at 2-8℃ for preservation.

[0069] Long-term culture preservation: preserved in the ultra-low temperature refrigerator by glycerol preservation method.

[0070] Example 2

[0071] This example is used to illustrate the antibacterial activity determination test of Priestia aryabhattai YN2302 on banana fusarium wilt pathogen.

[0072] The routine confrontation culture method was used to determine the antibacterial activity of YN2302 strain with banana fusarium wilt pathogen No. 4 physiological race Foc (referred to as TR4) as the indicator bacteria. The banana fusarium wilt pathogen is Fusarium oxysporum f. sp. cubense (Foc) No. 4 physiological race strain TR4 (15-1), which is isolated and preserved by the banana research team of the Institute of Agricultural Environment and Resources of Yunnan Academy of Agricultural Sciences. The banana fusarium wilt pathogen culture medium is PDA medium.

[0073] The plate confrontation method was used to determine the antibacterial rate of the strain: the banana fusarium wilt pathogen was transferred to a PDA plate (a plate with PDA medium), and cultured at 28℃ for 7 days. A sterile puncher was used to take a 5 mm diameter fungus cake along the edge of the colony, and the fungus cake was inoculated in the center of the PDA plate. Then, the YN2302 strain of Priestia aryabhattai was inoculated on the edge of the PDA plate with a distance of 25 mm from the center with a inoculation ring, and 4 points were inoculated symmetrically in each dish (see Figure 1 ). The above treatment was repeated 3 times, and the PDA plate inoculated with the pathogen only was used as a control (see Figure 2 ). After 7 days of culture at 28℃, the antibacterial effect was observed, and the diameter of banana fusarium wilt pathogen TR4 (15-1) was measured by cross method.

[0074] Antibacterial rate (%) = (control pathogen colony diameter - treated pathogen colony diameter) / control pathogen colony diameter x 100.

[0075] Results: The diameters of the treated pathogen colonies were 2.60 cm, 2.50 cm and 2.60 cm respectively, and the diameters of the control pathogen colonies were 8.90 cm, 8.90 cm and 8.80 cm respectively. The antibacterial rates of the three repeated experiments were 70.79%, 70.91% and 70.45% respectively, and the average antibacterial rate of Priestia aryabhattai on banana fusarium wilt pathogen was 70.72%. It has strong inhibition effect on the growth of banana fusarium wilt pathogen.

[0076] Example 3

[0077] This example is used to illustrate the effect of B. aryabhattai on the prevention and treatment of banana fusarium wilt and the growth promotion.

[0078] The preparation method of banana seedlings for the test: In the greenhouse, the tissue-cultured Brazilian banana seedlings (tissue-cultured and seedling-raised by the Banana Research Room of the Institute of Agricultural Environmental Resources of the Yunnan Academy of Agricultural Sciences) were washed to remove the root medium, then transplanted into seedling bags, and after the banana seedlings grew 3-4 leaves (about 1 month), they were transplanted into plastic pots with a diameter of 11 cm and a height of 12 cm with vermiculite as the substrate. After transplanting, water was often sprayed to keep the soil moist, and fertilizer was applied once a week (2 g of compound fertilizer per plant was dissolved in water and applied, the compound fertilizer contained N-P2O5-K2O at a mass fraction of 15-15-15, 1-2 times). After the banana seedlings grew 5-6 leaves (about 1 month), they were ready for use. 5

[0079] Preparation of B. aryabhattai fermentation broth: The ultra-low temperature preserved strain YN2302 was inoculated on a solid NA medium plate, incubated at 37°C in a constant temperature incubator for 1 day, and after a single colony grew, a sterile inoculation loop was used to pick a single colony and inoculate it into NA liquid medium, which was incubated at 37°C with shaking at 250 r / min for 3 days to obtain the strain fermentation broth. Sterile water was used to prepare the antagonistic bacteria fermentation broth with a concentration of 1×10 8 CFU / mL for standby.

[0080] Preparation of banana fusarium wilt spore solution: The 4th race cake of banana fusarium wilt bacteria after isolation and purification was inoculated into liquid PDA medium, which was incubated at 28°C with shaking at 250 r / min for 3 days, and the culture solution was filtered with 4 layers of sterile gauze to obtain a pathogenic bacteria spore suspension. Sterile water was used to prepare the banana fusarium wilt bacteria spore solution with a concentration of 1×10 6 CFU / mL for standby, and the banana fusarium wilt bacteria spore solution is referred to as TR4.

[0081] (I) Banana fusarium wilt potting prevention and treatment effect test:

[0082] The test was set up with 2 treatments: treatment group 1: irrigation with NA liquid culture solution + banana fusarium wilt spore solution (CK+TR4); treatment group 2: irrigation with B. aryabhattai fermentation broth + banana fusarium wilt spore solution (YN2302+TR4). Each treatment group had 3 replicates, and each replicate had 10 banana seedlings.

[0083] Test method: The banana seedlings with consistent growth were irrigated with the antagonistic bacteria fermentation broth (B. aryabhattai YN2302: concentration of 1×10 8 CFU / mL) at the root, 40 mL per plant, and the control was irrigated with 40 mL of NA liquid culture solution per plant. After 7 days, the banana seedlings were irrigated with banana fusarium wilt spore solution (TR4) with a concentration of 1×10​6 CFU / mL of F. oxysporum spore solution was irrigated on the roots of potted banana plants, 40 mL per plant, and the NA liquid culture was irrigated as a control.

[0084] After 45 days of irrigating F. oxysporum spore solution, the disease incidence of treatment groups 1 and 2 was investigated, the disease index of each treatment group was counted, and the control effect was calculated.

[0085] Table 1 is the grading standard for banana wilt disease.

[0086] Table 1

[0087]

[0088]

[0089]

[0090] (II) Banana plant growth promotion test:

[0091] Treatment group 3: irrigate NA liquid culture (CK); treatment group 4: irrigate Bacillus aryabhattai fermentation liquid (YN2302). Two treatment groups were set up with 3 replicates, and 10 banana seedlings per replicate. The fermentation liquid of the strain resistant to banana wilt (Bacillus aryabhattai YN2302: concentration 1×10 8 CFU / mL) was irrigated on the roots of the above-mentioned potted banana plants with consistent growth, 40 mL per plant, and 40 mL of NA liquid culture was irrigated per plant as a control.

[0092] On the day of irrigating Bacillus aryabhattai fermentation liquid, the plant height, pseudostem diameter, leaf number and other bioinformatics indicators of treatment groups 3 and 4 (0 dpi CK and 0 dpi YN2302) banana plants were measured; 45 days after irrigating Bacillus aryabhattai fermentation liquid, the plant height, pseudostem diameter, leaf number, aboveground fresh weight, underground fresh weight and other bioinformatics indicators of treatment groups 3 and 4 (45 dpi CK and 45 dpi YN2302) banana plants were measured, and the growth promotion effect of Bacillus aryabhattai on banana plants was calculated.

[0093] Measurement method:

[0094] Measurement and recording method: plant height (measure the distance from the ground to the intersection point of the top two leaf petioles);

[0095] Pseudostem diameter: measure the diameter of the pseudostem base about 1 cm from the ground with a vernier caliper;

[0096] Aboveground fresh weight: the weight of the pseudostem above the ground about 1 cm from the ground;

[0097] Underground fresh weight: the weight of the banana seedling about 1 cm below the ground.

[0098] Leaf number: the number of green leaves of each plant was recorded;

[0099] Leaf length and width: the length and width of the first expanded leaf were measured.

[0100] Results:

[0101] 1. Potting control effect:

[0102] (1) Leaf disease index and control effect: as shown in Table 2 and Figure 3 Table 3, the leaf disease index of the banana wilt treated with Bacillus aryabhattai fermentation liquid for 3 times was 27.50, 35.00 and 20.00, with an average of 27.50, and the leaf disease index of the control for 3 times was 62.50, 57.50 and 62.50, with an average of 60.83. The potting control effect of the leaf was 56.00%, 39.13% and 68.00%, with an average of 54.38%. Figure 3 It can also be seen that after the plant was irrigated with the fermentation liquid containing Bacillus aryabhattai, the leaf grew well and the color was emerald green.

[0103] (2) Corm disease index and control effect: as shown in Table 2 and Figure 3 Table 3, the corm disease index of the banana wilt treated with Bacillus aryabhattai fermentation liquid for 3 times was 12.50, 12.50 and 5.00, with an average of 10.00, and the leaf disease index of the control for 3 times was 40.00, 42.50 and 55.00, with an average of 45.83. The potting control effect of the corm was 68.75%, 70.59% and 90.91%, with an average of 76.75%. Figure 3 It can also be seen that the corm of the plant not irrigated with the fermentation liquid containing Bacillus aryabhattai was severely damaged and had obvious black-brown color, while the corm of the plant irrigated with the fermentation liquid of Bacillus aryabhattai was healthy and almost had no black-brown color.

[0104] Table 2

[0105]

[0106] 2. Promoting effect on the plant:

[0107] After 45 days of treatment with Bacillus aryabhattai fermentation liquid, it can be seen that compared with the control, the banana plant treated with Bacillus aryabhattai fermentation liquid had obvious differences in the biological information indexes such as plant height, leaf number, pseudostem diameter, aboveground and underground fresh weight, and the specific differences were as follows: Figure 4

[0108] ​(1) Promoting effect on leaf number

[0109] The leaf number of the control CK (0 dpi CK) was 5.10 and the leaf number of the treatment YN2302 (0 dpi YN2302) was 5.20 on the day of watering the Bacillus aryabhattai fermentation broth. The leaf number of the control CK (45 dpi CK) was 5.33 and the leaf number of the treatment YN2302 (45 dpi YN2302) was 6.07 after 45 days of watering the Bacillus aryabhattai fermentation broth.

[0110] Result analysis: The leaf number of the treatment after 45 days of watering the Bacillus aryabhattai (45 dpi YN2302) was significantly different from the control after 45 days (45 dpi CK), the treatment on the day of watering the antagonistic bacteria (0 dpi YN2302) and the control treatment on the day of watering the antagonistic bacteria (0 dpi CK). However, the control after 45 days (45 dpi CK) was not significantly different from the treatment on the day of watering the antagonistic bacteria (0 dpi YN2302) and the control treatment on the day of watering the antagonistic bacteria (0 dpi CK). This shows that the Bacillus aryabhattai fermentation broth has a significant promoting effect on the leaf number of banana plants.

[0111] (2) Promoting effect on plant height

[0112] The plant height of the treatment CK (0 dpi CK) was 16.87 cm and the plant height of the treatment YN2302 (0 dpi YN2302) was 17.56 cm on the day of watering the Bacillus aryabhattai fermentation broth. The plant height of the control CK (45 dpi CK) was 29.86 cm and the plant height of the treatment YN2302 (45 dpi YN2302) was 35.71 cm after 45 days of watering the Bacillus aryabhattai fermentation broth.

[0113] Result analysis: The plant height of the treatment after 45 days of watering the Bacillus aryabhattai (45 dpi YN2302) was significantly different from the control after 45 days (45 dpi CK), the treatment on the day of watering the antagonistic bacteria (0 dpi YN2302) and the control treatment on the day of watering the antagonistic bacteria (0 dpi CK). This shows that the Bacillus aryabhattai fermentation broth has a significant promoting effect on the plant height of banana plants.

[0114] (3) Promoting effect on the thickness of the pseudostem

[0115] The diameter of the false stem of the CK (0 dpi CK) was 1.33 cm, and the diameter of the false stem of the treatment YN2302 (0 dpi YN2302) was 1.37 cm on the day of watering the Bacillus aryabhattai fermentation liquor. After 45 days of watering the Bacillus aryabhattai fermentation liquor, the diameter of the false stem of the CK (45 dpi CK) was 1.68 cm, and the diameter of the false stem of the treatment YN2302 (45 dpi YN2302) was 1.77 cm.

[0116] Result analysis: The diameter of the false stem of the treatment (45 dpi YN2302) after 45 days of watering the Bacillus aryabhattai was higher than that of the control (45 dpi CK) after 45 days, the treatment (0 dpi YN2302) on the day of watering the antagonistic bacteria, and the control treatment (0 dpi CK) on the day of watering the antagonistic bacteria. It shows that the Bacillus aryabhattai fermentation liquor has a promoting effect on the diameter of the banana plant false stem.

[0117] (4) Effect on the fresh weight of the aboveground part of banana plants

[0118] After 45 days of watering the Bacillus aryabhattai, the fresh weight of the aboveground part of the CK (45 dpi CK) was 28.47 g, and the fresh weight of the aboveground part of the treatment YN2302 (45 dpi YN2302) was 32.43 g.

[0119] Result analysis: There was a significant difference in the fresh weight of the aboveground part of the treatment (45 dpi YN2302) after 45 days of watering the Bacillus aryabhattai and the control (45 dpi CK) after 45 days.

[0120] (5) Effect on the fresh weight of the underground part of banana plants

[0121] After 45 days of watering the Bacillus aryabhattai, the fresh weight of the underground part of the CK (45 dpi CK) was 32.53 g, and the fresh weight of the underground part of the treatment YN2302 (45 dpi YN2302) was 35.67 g.

[0122] Result analysis: There was a significant difference in the fresh weight of the underground part of the treatment (45 dpi YN2302) after 45 days of watering the Bacillus aryabhattai and the control (45 dpi CK) after 45 days.

[0123] (6) Effect on leaf length

[0124] The length of the first unfolded leaf of the treatment CK (0 dpi CK) was 18.13 cm, and the length of the first unfolded leaf of the treatment YN2302 (0 dpi YN2302) was 18.54 cm on the day of watering the Bacillus aryabhattai fermentation liquor. After 45 days of watering the Bacillus aryabhattai fermentation liquor, the length of the first unfolded leaf of the treatment CK (45 dpi CK) was 18.73 cm, and the length of the first unfolded leaf of the treatment YN2302 (45 dpi YN2302) was 19.42 cm.

[0125] Result analysis: The length of the first unfolded leaf of the treatment (45 dpi YN2302) after 45 days of watering the Bacillus aryabhattai fermentation liquor was higher than that of the control (45 dpi CK) after 45 days, the treatment (0 dpi YN2302) on the day of watering the antagonistic bacteria, and the control treatment (0 dpi CK) on the day of watering the antagonistic bacteria. This shows that the Bacillus aryabhattai fermentation liquor has a certain effect on the leaf length of banana plants.

[0126] (7) Effect on leaf width

[0127] The width of the first unfolded leaf of the treatment CK (0 dpi CK) was 7.63 cm, and the width of the first unfolded leaf of the treatment YN2302 (0 dpi YN2302) was 7.9 cm on the day of watering the Bacillus aryabhattai fermentation liquor. After 45 days of watering the Bacillus aryabhattai fermentation liquor, the width of the first unfolded leaf of the treatment CK (45 dpi CK) was 8.16 cm, and the width of the first unfolded leaf of the treatment YN2302 (45 dpi YN2302) was 8.05 cm.

[0128] Result analysis: The width of the first unfolded leaf of the treatment (45 dpi YN2302) after 45 days of watering the Bacillus aryabhattai fermentation liquor was higher than that of the control (45 dpi CK) after 45 days, the treatment (0 dpi YN2302) on the day of watering the antagonistic bacteria, and the control treatment (0 dpi CK) on the day of watering the antagonistic bacteria. This shows that the Bacillus aryabhattai fermentation liquor has a certain effect on the leaf width of the first unfolded leaf of banana plants.

[0129] Table 3

[0130]

[0131] Note: The data is the average value ± standard error, and the lower case letters indicate a 5% significant difference between the two groups.

[0132] The results of the pot experiment show that the Bacillus aryabhattai fermentation liquor has no inhibitory effect on the growth of banana plants and has a significant growth-promoting effect on the plant height, leaf number, pseudostem diameter, fresh weight of the aboveground and underground parts, and other aspects of banana plants.

[0133] The above experimental data show that the strain is a functional bacteria with disease prevention and growth promotion effect, can inhibit the invasion and harm of banana fusarium wilt pathogen to banana plants, and also has a significant growth promotion effect on banana plants. The bacillus aryabhattai provided by the application can provide excellent strain resources for biological growth promotion and disease resistance, and can also provide a reference for the development of biological products such as biological fertilizer and biological agent for banana fusarium wilt.

[0134] The preferred embodiments of the application are described in detail above, but the application is not limited thereto. Within the technical concept of the application, various simple modifications can be made to the technical solutions of the application, including the combination of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as disclosed by the application and fall within the protection scope of the application.

Claims

1. A strain of Priestia aryabhattai, characterized in that, The Bacillus aryabhattai has a preservation number of CCTCC NO: M 2024282.

2. An inoculant characterized in that, The bacterial agent contains the Bacillus aryabhattai according to claim 1.

3. The Bacillus aryabhattai according to claim 1 or the bacterial agent according to claim 2 is used for inhibiting Fusarium oxysporum f. sp. cubense. and / or, 4 x 10 7 CFU Fusarium oxysporum f. sp. cubense, the amount of B. aurescens is not less than 1 x 10 8 CFU, preferably 8 x 10 8 CFU to 1 x 10 10 CFU, more preferably 1 x 10 9 CFU to 6 x 10 9 CFU.

4. A method of controlling banana fusarium wilt and / or promoting the growth of banana plants, characterized by, The method comprises applying the fermentation liquor of the Bacillus aryabhattai according to claim 1 or the bacterial agent according to claim 2 to banana plants.

5. The method of claim 4, wherein, The preparation method of the fermentation liquor comprises inoculating the Bacillus aryabhattai into a liquid culture medium for culture.

6. The method of claim 4 or 5, wherein, The liquid culture medium contains yeast extract, peptone, beef extract, glucose and water. Preferably, the weight ratio of the yeast extract, peptone, beef extract and glucose is 1:(5-14):(1-5):(8-25). Preferably, the total weight of the yeast extract, peptone, beef extract and glucose accounts for 1-5% of the weight of the culture medium, more preferably 2-3%.

7. The method of claim 6, wherein, The total nitrogen content in the dry base of the yeast extract is above 5% by weight. Preferably, the sodium chloride content in the dry base of the yeast extract is below 6% by weight.

8. The method of claim 5, wherein, The pH of the liquid culture medium is 6-8.

9. The method of claim 5, wherein, The culture conditions comprise a time of 2-5 days and a temperature of 35-40℃.

10. The method of claim 4 or 5, wherein, The Bacillus aryabhattai is administered in an amount of not less than 1 x 10 8 CFU / strain / time, preferably 8 x 10 8 CFU / strain / time, preferably 8 x 10 10 CFU / strain / time, preferably 8 x 10 9 CFU / strain / time, preferably 8 x 10 9 CFU / strain / time; or The amount of the fermentation broth of the bacterial agent is such that the application amount of Bacillus aryabhattai is not less than 1 x 10 8 CFU / strain / time, preferably 8 x 10 8 CFU / strain / time to 1 x 10 10 CFU / strain / time, more preferably 1 x 10 9 CFU / strain / time to 6 x 10 9 CFU / strain / time.

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